Diaminofluorescein derivatives

ABSTRACT

A compound represented by the following formula (I):  
                 
 
     wherein R 1  and R 2  represent amino groups that substitute at adjacent positions on the phenyl ring, provided that either of R 1  and R 2  represents a mono(C 1-6  alkyl)-substituted amino group and the other represents an unsubstituted amino group; and R 3  and R 4  independently represent hydrogen atom or an acyl group, and an agent for measurement of nitrogen monoxide which comprises said compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.10/140,059, filed May 8, 2002, which is a continuation of applicationSer. No. 09/487,830, filed Jan. 20, 2000. The entire disclosures ofapplication Ser. Nos. 10/140,059 and 09/487,830 are considered as beingpart of the disclosure of this application, and the entire disclosure ofapplication Ser. Nos. 10/140,059 and 09/487,830 are expresslyincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to fluorescein derivatives whichare useful as agents for measurement of nitrogen monoxide. It alsorelates to agents for measuring nitrogen monoxide which comprise saidcompound.

RELATED ART

[0003] Nitrogen monoxide (NO) is an unstable radical having a shortlife, and it has been elucidated that nitrogen monoxide has importantfunctions as a physiologically active substance in a living body(featured in Gendai Kagaku (Chemistry Today), April, 1994). Methods formeasuring nitrogen monoxide are mainly classified into indirect methodswhere oxidative degradation products of nitrogen monoxide such as NO₂ ⁻or NO₃ ⁻ are measured, and methods where nitrogen monoxide is directlymeasured. The direct methods have been focused from the standpoint thatthey achieve detection and quantification of nitrogen monoxide underphysiological condition. However, no measuring method has been developedto date that has sufficient specificity and high sensitivity and isapplicable to an in vitro system.

[0004] For example, a chemiluminescence method which utilizesluminescence emitted during an ozonic oxidation of NO radicals (Palmer,R. M., et al., Nature, 327, pp.524-526, 1987); a method which comprisesthe step of measuring an absorption spectrum of metHb that is producedby an oxidation of oxyhemoglobin (O₂Hb) (Kelm, M., et al., Circ. Res.66,pp.1561-1575, 1990); a method which comprises the step of measuringelectric current generated during an oxidation by means of electrodesthat are inserted into a tissue (Shibuki, K., Neurosci. Res. 9,pp.69-76, 1990; Malinski, T., Nature, 356, pp.676-678, 1992); and theGriess reaction method (Green, L. C., et al., Anal. Biochem., 126,pp.131-138, 1992) are known as typical methods (as reviews, see, “3.Method for measurement of NO,” by Tetsuo Nagano, pp.42-52, “Approachfrom the Latest Medicine 12, NO” edited by Noboru Toda, published byMedical View Co., Ltd.; and Archer, S., FASEB J., 7, pp.349-360, 1993).

[0005] The Griess reaction method comprises a detection step thatutilizes azo coupling between naphthylethylenediamine and a diazoniumsalt compound formed with NO₂ ⁻ which is generated by the oxidation ofnitrogen monoxide radicals. This method is advantageous because it doesnot require particular apparatuses or techniques, although nitrogenmonoxide radicals are not directly measured by the method. In addition,NO₃ ⁻ can also be measured after being reduced to NO₂ ⁻ by using cadmium(Stainton, M. P., Anal. Chem., 46, p.1616, 1974; Green, L. C., et al.,Anal. Biochem., 126, pp.131-138, 1982) or hydrazine (Sawicki, C. R. andScaringelli, F. P., Microchem. J., 16, pp.657-672, 1971), andaccordingly, the method also has characteristic feature that it enablesthe measurement of metabolites related to nitrogen monoxide.

[0006] 2,3-Diaminonaphthalene has also been known as an agent formeasuring nitrogen monoxide by detecting NO₂ ⁻, as in a similar mannerto Griess reaction method. This agent reacts with NO₂ ⁻ under an acidiccondition to form a fluorescent adduct, i.e., naphthalenetriazole(chemical name: 1-[H]-naphtho[2,3-d]triazole) (Wiersma, J. H., Anal.Lett., 3, pp.123-132, 1970). Details of the reaction conditions of2,3-diaminonaphthalene and NO₂ ⁻ have been studied, and it has beenfound that the reaction proceeds most rapidly at a pH not higher than 2,and completes in about 5 minutes at room temperature (Wiersma, J. H.,Anal. Lett., 3, pp.123-132, 1970; Sawicki, C. R., Anal. Lett., 4,pp.761-775, 1971). The resulting adduct emits fluorescence mostefficiently at a pH not lower than 10 (Damiani, P. and Burini, G.,Talanta, 8, pp.649-652, 1986).

[0007] The method for measuring nitrogen monoxide using the above2,3-diaminonaphthalene has characteristic features of 50- to 100-foldhigher sensitivity compared to the Griess reaction method, since itsdetection limit is as low as approximately several tens nM (Misko, T.P., Anal. Biochem. 214, pp.11-16, 1993). This method is highlyadvantageous because it needs no particular apparatus or technique andcan be carried out conveniently (as a review of the aforementionedmethod, see, DOJIN News. No. 74, Information, “An agent for thedetermination of NO: 2,3-diaminonaphthalene,” Dojindo Laboratories Inc.,1995). However, the method does not utilize nitrogen monoxide, per se,but utilizes an oxidation product, i.e., NO₂ ⁻, as a reactant.Accordingly, the method is considered as an indirect method whencompared to those including direct measurement of nitrogen monoxide.Furthermore, because the reaction of 2,3-diaminonaphthalene with NO₂ ⁻is carried out under a strongly acidic condition (pH not higher than 2),the method has a problem in that it cannot be employed for detection orquantification of nitrogen monoxide under a physiological condition.

[0008] The inventors of the present invention conducted researches toprovide a means that enables direct and highly sensitive measurement ofnitrogen monoxide under a physiological condition, and as a result, theyfound that nitrogen monoxide can efficiently react with2,3-diaminonaphthalene or its derivatives, even under a neutralcondition, in the presence of an oxygen source such as dissolved oxygenor oxide compounds (e.g., PTIO and derivatives thereof such ascarboxy-PTIO), and a fluorescent naphthalenetriazole or a derivativethereof is obtained. They also found that a method for measuringnitrogen monoxide utilizing the above reaction has extremely highdetection sensitivity, and can achieve accurate quantification of verysmall amount of nitrogen monoxide (see, the specification of JapanesePatent Application No. Hei 7-189978).

[0009] However, the aforementioned method utilizing2,3-diaminonaphthalene requires the irradiation with excitation lighthaving a short wavelength of approximately 370-390 nm for the detectionof fluorescence, and this may cause damages to cells and/or tissues in ameasurement system. Strong autofluorescence of cells, per se, may alsopossibly affect the measurement, and moreover, there is a problem that afluorescence filter provided on a usual fluorescence microscope fails tosufficiently cut off excitation light during fluorescence measurement.In addition, the fluorescent triazole compound formed from2,3-diaminonaphthalene has rather insufficient fluorescence intensity,and therefore, it is difficult to achieve accurate measurement ofintracellular fluorescence of an individual cell by ordinaryfluorescence microscopy. Since 2,3-diaminonaphthalene itself has asimple chemical structure, there is also a problem that the compound isnot suitable as a fundamental structure for various chemicalmodifications so as to achieve intracellular localization of an agent.

[0010] As a method for measuring nitrogen monoxide that solved theseproblems, the inventors of the present invention proposed a methodutilizing a class of diaminofluorescein derivatives (U.S. Pat. No.5,874,590). By using the derivatives, nitrogen monoxide can be measuredwith an excitation light having a long wavelength that gives no damageto living tissues or cells, and intracellularly existing nitrogenmonoxide can be accurately measured for each individual cell. Thediaminofluorescein derivatives are extremely satisfactory agents fromthe standpoints of reactivity with nitrogen monoxide and measurementsensitivity. However, the derivatives have a problem that thefluorescence intensity of the triazole derivatives produced through thereaction with nitrogen monoxide slightly increases in the weakly basicto weakly acidic region. Moreover, the triazole compounds also have aproblem of instability to light. For these reasons, it has been desiredto develop an agent for measurement of nitrogen monoxide that gives nochange in fluorescence intensity due to alteration in pH, and produces afluorescent substance that is stable to light.

DISCLOSURE OF THE INVENTION

[0011] An object of the present invention is to provide compounds whichare useful for measurement of nitrogen monoxide. More specifically, theobject of the present invention is to provide compounds that enable themeasurement of nitrogen monoxide by means of excitation light having along wavelength which does not cause damages to living tissues andcells, and can efficiently react with nitrogen monoxide under a neutralcondition to provide a fluorescent substance having excellentfluorescence intensity, which fluorescent substance has stability tolight and whose fluorescence intensity is not affected by alteration inpH.

[0012] Another object of the present invention is to provide an agentfor measuring nitrogen monoxide which comprises a compound having theaforementioned characteristic features. More specifically, the object isto provide an agent for measuring nitrogen monoxide which enablesaccurate measurement of intracellularly existing nitrogen monoxide forindividual cells.

[0013] The inventors of the present invention made efforts to achievethe foregoing objects, and as a result, they found that a particularclass of fluorescein derivatives, which themselves emit almost nofluorescence, can easily react with nitrogen monoxide under a neutralcondition, and give triazole compounds having high fluorescenceintensity. They also found that the triazole derivatives can emit strongfluorescence at approximately 515 nm when irradiated with excitationlight having a longer wavelength of about 495 nm, and that thederivatives gave substantially no change in fluorescence intensity fromweakly basic to weakly acidic regions and the derivatives were stable tolight. They further found that intracellular nitrogen monoxideconcentrations in individual cells were accurately and convenientlymeasurable by using these compounds as an agent for measuring nitrogenmonoxide. The present invention was achieved on the basis of thesefindings.

[0014] The present invention thus provides a compound represented by thefollowing formula (I):

[0015] wherein R¹ and R² represent amino groups that substitute atadjacent positions on the phenyl ring, provided that either of R¹ and R²represents a mono(C₁₋₆ alkyl)-substituted amino group and the otherrepresents an unsubstituted amino group; and R³ and R⁴ independentlyrepresent hydrogen atom or an acyl group.

[0016] According to preferred embodiments of the present invention,there are provided the aforementioned compound wherein themonoalkyl-substituted amino group represented by either of R¹ and R² isa monomethylamino group; and the aforementioned compound wherein both ofR³ and R⁴ represent hydrogen atom.

[0017] According to another aspect of the present invention, there isprovided an agent for measurement of nitrogen monoxide which comprisesthe aforementioned compound.

[0018] According to further aspect of the present invention, there isprovided a compound represented by the following formula (II):

[0019] wherein R¹¹ and R¹² combine together to form a group representedby —N═N—N(R¹⁹)— which forms a ring structure at adjacent positions onthe phenyl ring, wherein R¹⁹ represents a C₁₋₆ alkyl group, or R¹¹ andR¹² represent a combination of an amino group and a nitro group whichsubstitute at adjacent positions on the phenyl ring; and R¹³ and R¹⁴independently represent hydrogen atom or an acyl group.

[0020] The present invention further provides a method for measuringnitrogen monoxide which comprises the steps of:

[0021] (1) reacting a compound represented by the above formula (I) withnitrogen monoxide; and

[0022] (2) detecting a compound of the formula (II) formed by the abovestep (1).

BRIEF EXPLANATION OF THE DRAWINGS

[0023]FIG. 1 shows changes in fluorescence intensity of the triazolederivatives disclosed in U.S. Pat. No. 5,874,590 (DAF-2T and DAF-5T) andDAF-FM T due to alteration in pH.

[0024]FIG. 2 shows photostability of the triazole derivatives disclosedin U.S. Pat. No. 5,874,590 (DAF-1T, DAF-2T, DAF-4T and DAF-5T) andDAF-FM T.

[0025]FIG. 3 shows results of measurement of nitrogen monoxide formedextracellularly by bovine aorta endothelial cells.

[0026]FIG. 4 shows results of measurement of nitrogen monoxide formedintracellularly by bovine aorta endothelial cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0027] In the above general formula (I), R¹ and R² represent aminogroups which substitute at adjacent positions on the phenyl ring. Eitherof R¹ and R² represents a mono(C₁₋₆ alkyl)substituted amino group, andthe other represents an unsubstituted amino group. The C₁₋₆ alkyl groupconstituting the mono(C₁₋₆ alkyl)substituted amino group may be straightor branched. More specifically, methyl group, ethyl group, n-propylgroup, isopropyl group, n-butyl group, sec-butyl group, tert-butyl groupand the like may be used. Other C₁₋₆ alkyl groups or C₁₋₆ alkyl moietiesof functional groups containing a C₁₋₆ alkyl moiety, which are referredto in the specification, may be similar to those explained above.

[0028] R³ and R⁴ independently represent hydrogen atom or an acyl group.Examples of the acyl group include, for example, an arylcarbonyl groupsuch as benzoyl group, p-methoxybenzoyl group, p-chlorobenzoyl group, ornaphthylcarbonyl group; a C₁₋₆ alkylcarbonyl group such as acetyl group,propionyl group, or butanoyl group and the like. Preferably, R³ and R⁴are independently hydrogen atom or an acetyl group, and most preferably,both of R³ and R⁴ are hydrogen atoms or both are acetyl groups.

[0029] In the aforementioned formula (II), R¹¹ and R¹² combine togetherto represent the group —N═N—N(R¹⁹)— which forms a ring structure atadjacent positions on the phenyl ring. R¹⁹ represents a C₁₋₆ alkylgroup. R¹¹ and R¹² also represent a combination of an amino group and anitro group which substitute at adjacent positions on the phenyl ring,wherein either of R¹¹ and R¹² represents an amino group and the otherrepresents a nitro group. The amino group represented by R¹¹ or R¹² maybe unsubstituted, or may have one C₁₋₆ alkyl group. The amino group mayhave an acyl group such as acetyl group, trifluoroacetyl group, orbenzoyl group, or a protective group such as alkylsilyl groups includingtrimethylsilyl group. An arylalkyl group such as benzyl group may alsobe used as the protective group.

[0030] R¹³ and R¹⁴ independently represent hydrogen atom or an acylgroup. As the acyl group, for example, an arylcarbonyl group such asbenzoyl group, p-methoxybenzoyl group, p-chlorobenzoyl group, ornaphthylcarbonyl group; a C₁₋₆ alkylcarbonyl group such as acetyl group,propionyl group, or butanoyl group and the like may be used. Preferably,R¹³ and R¹⁴ independently represent hydrogen atom or acetyl group, andmost preferably, both of R¹³ and R¹⁴ are hydrogen atoms or acetylgroups.

[0031] The compounds of the formula (I) and the formula (II) wherein R¹¹and R¹²represent the combination of an amino group and a nitro groupsubstituting at adjacent positions on the phenyl ring can be prepared,for example, according to the methods described in U.S. Pat. No.5,874,590, and the details of the methods will be specifically explainedin the example section of the specification. It will be understood thatthe compounds of the formula (II) are useful as synthetic intermediatecompounds for the preparation of the compounds of the formula (I). Amongthe compounds represented by the formula (II), those wherein R¹¹ and R¹²combine together to represent the group —N═N—N(R¹⁹)— that forms a ringstructure at adjacent positions on the phenyl ring can be prepared byreacting the compounds of the aforementioned formula (I) with nitrogenmonoxide. These compounds are highly fluorescent as explained later, andare useful for the measurement of nitrogen monoxide.

[0032] By referring to the methods for preparation of the fluoresceinderivatives described in the aforementioned publication and specificexplanations in the examples, one of ordinarily skilled artisan willreadily understand that the compounds falling within the scope of theformula (I) and the formula (II) can easily be prepared. Methods forpreparing fluorescein derivatives having variety of substituents areknown, and therefore, those skilled in the art can readily prepare anycompounds that fall within the formula (I) and (II) by combining knownmethods available to skilled artisan with the methods disclosed in theexamples of the specification.

[0033] The compounds of the formula (I) and the formula (II) accordingto the present invention may have one or more asymmetric carbon atoms.Any optical isomers of the compounds based on one or more asymmetriccarbon atoms which are optically pure forms, any mixtures of the opticalisomers, racemates, diastereoisomers in pure forms, mixtures of thediastereoisomers and the like fall within the scope of the presentinvention. The compounds of the formula (I) and the formula (II) of thepresent invention may exist as base addition salts such as sodium saltsor potassium salts, or acid addition salts such as hydrochlorides,sulfates, or p-toluenesulfonates. Any one of these salts also fallswithin the scope of the present invention. Furthermore, the compounds ofthe present invention in free form or in the form of a salt may exist ashydrates or solvates, and it should be understood that they also fallwithin the scope of the present invention, and they can be utilized asthe agent for measurement of the present invention.

[0034] Fluorescein derivatives are known to exist also as compoundswithout the formation of a lactone ring, i.e.,9-(o-carboxyphenyl)-6-hydroxy-3H-xanthen-3-one derivatives. Thecompounds of the present invention may also exist in the form of theaforementioned structural isomer, and it will be readily understood bythose skilled in the art that they also fall within the scope of thepresent invention. In the formulas (I) and (II), and in the schemes setout above, only compounds having a lactone ring are shown forconvenience.

[0035] Thus, the present invention is also directed to a compoundrepresented by the following formula (I′):

[0036] wherein R¹ and R² represent amino groups that substitute atadjacent positions on the phenyl ring, provided that either of R¹ and R²represents a mono(C₁₋₆ alkyl)-substituted amino group and the otherrepresents an unsubstituted amino group and wherein R³ representshydrogen atom or an alkyl group and R⁴ represents hydrogen atom or anacyl group. Moreover, the monoalkyl-substituted amino group representedby either of R¹ and R² can be a monomethylamino group, and both of R³and R⁴ can represent hydrogen atoms.

[0037] Moreover, the present invention is also directed to a compoundrepresented by the following formula (II′):

[0038] wherein R¹¹ and R¹² combine together to form a group representedby —N═N—N(R¹⁹)— which forms a ring structure at adjacent positions onthe phenyl ring, wherein R¹⁹ represents a C₁₋₆ alkyl group, or R¹¹ andR¹² represent a combination of an amino group and a nitro group whichsubstitute at adjacent positions on the phenyl ring; and R¹³ representshydrogen atom or an alkyl group, and R¹⁴ represents hydrogen atom or anacyl group.

[0039] Still further, the present invention is directed to a method formeasuring nitrogen monoxide which comprises:

[0040] reacting a compound represented by formula (I′) with nitrogenmonoxide to form a compound represented by formula (II′);

[0041] irradiating the compound represented by formula (II′) with lightto cause fluorescence of the irradiated compound; and

[0042] measuring the fluorescence;

[0043] wherein the compound represented by formula (I′) has thefollowing formula:

[0044] wherein R¹ and R² represent amino groups that substitute atadjacent positions on the phenyl ring, provided that either of R¹ and R²represents a mono(C₁₋₆ alkyl)-substituted amino group and the otherrepresents an unsubstituted amino group and wherein R³ representshydrogen atom or an alkyl group and R⁴ represents hydrogen atom or anacyl group; and

[0045] wherein the compound represented by formula (II′) has thefollowing formula:

[0046] wherein R¹¹ and R¹² combine together to form a group representedby —N═N—N(R¹⁹)— which forms a ring structure at adjacent positions onthe phenyl ring, wherein R¹⁹ represents a C¹⁻⁶ alkyl group, or R¹¹ andR¹² represent a combination of an amino group and a nitro group whichsubstitute at adjacent positions on the phenyl ring; and R¹³ representshydrogen atom or an alkyl group, and R¹⁴ represents hydrogen atom or anacyl group.

[0047] The compounds represented by the formula (I) of the presentinvention have a characteristic property that they efficiently reactwith nitrogen monoxide under a neutral condition and provide compoundsof the formula (II) wherein R¹¹ and R¹² combine together to form thegroup of —N═N—N(R¹⁹)— which forms a ring structure at adjacent positionson the phenyl ring. The compounds represented by the formula (I), perse, emit almost no fluorescence when irradiated with excitation light of495 nm under a neutral condition, whereas the compounds of the aboveformula (II) have a property of emitting extremely strong fluorescence(emission: 515 nm) under the same condition. Therefore, nitrogenmonoxide in a living tissue or a cell can be measured by introducing thecompound represented by the formula (I) into a living tissue or a cellto allow the compound react with nitrogen monoxide to form thefluorescent compound represented by the above formula (II), and thenmeasuring fluorescence of said compound. The compounds of the formula(II) formed as described above have an excellent property that they givesubstantially no change in fluorescence intensity from a weakly basicregion of about pH 9 to a weakly acidic region of about pH 6.

[0048] The method for measurement of nitrogen monoxide provided by thepresent invention comprises the steps of reacting a compound representedby the above formula (I) with nitrogen monoxide to form a compound offormula (II), and then measuring fluorescence of the compound of theformula (II). The term “measurement” used in the specification should beconstrued in its broadest sense, which includes measurements for varietyof purposes such as, for example, detection, quantification, qualitativeanalysis and the like. The above reaction can preferably be carried outunder a neutral condition, for example, in the range of from pH 6.0 to8.0, preferably in the range of from pH 6.5 to 7.8, and more preferablyin the range of from pH 6.8 to 7.6. However, the measurements ofnitrogen monoxide according to the present invention are not limited tothose conducted under the neutral range. For example, measurements canalso be performed under a strongly acidic condition such as in gastricmucosal cells.

[0049] The compounds wherein R³ and R⁴ are acetyl groups can easily passthrough cellular membranes so as to be taken intracellularly, and thenthey are converted into the compounds wherein R³ and R⁴ are hydrogenatoms after the hydrolysis of the ester of the acetoxy groups. Theresulting dihydroxy compound are highly hydrophilic, and not easilyexcreted extracellularly from the intracellular environment.Accordingly, the compound wherein R³ and R⁴ are acetyl groups are usefulas an agent for measurement, per se, but useful as a so-called pro-drugfor intracellularly transporting the measuring agent (the compoundwherein R³ and R⁴ are hydrogen atoms) at a high concentration.

[0050] The measurement of fluorescence can be carried out according to aknown fluorometry method (see, for example, Wiersma, J. H., Anal. Lett.,3, pp.123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp.761-775, 1971;Damiani, P. and Burini, G., Talanta, 8, pp.649-652, 1986; Damiani, P.and Burini, G., Talanta, 8, pp.649-652, 1986; and Misko, T. P., Anal.Biochem., 214, pp.11-16, 1993). For the nitrogen monoxide measurementaccording to the present invention, for example, irradiation with lightof about 495 nm as excitation light, and measurement of fluorescence ofabout 515 nm may preferably be performed. By using the light having suchwavelength, efficient cut off can be achieved by using a fluorescencefilter provided on an ordinary fluorescence microscope, and measurementwith high 67 can be performed without using a special filter.

[0051] Where particularly high sensitive measurement is required, theaforementioned measurement of nitrogen monoxide may be carried out inthe presence of an oxygen source. As the oxygen source, for example,oxygen, ozone, oxide compounds or the like can be used. As the oxygen,dissolved oxygen can generally be used, and if desired, oxygen gas maybe introduced into the reaction system or an agent that can generateoxygen (e.g., hydrogen peroxide) may be added. The oxide compounds arenot particularly limited so long as they have an oxide bond that caneasily be cleaved to release oxygen, e.g., N—O, S—O, or P—O. Forexample, PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,Maeda, H., et al., J. Leuk. Biol., 56, pp.588-592, 1994; and Akaike, T.,et al., Biochemistry, 32, pp.827-832, 1993) or derivatives thereof(carboxy-PTIO which has carboxyl group introduced at the para-positionof the phenyl group of PTIO), triphenylphosphine oxide, triethylamineoxide or the like can be used.

[0052] Among the oxide compounds mentioned above, PTIO and derivativesthereof (e.g., carboxy-PTIO) are particularly preferred compounds, andthey can be readily obtained by those skilled in the art (listed in, forexample, Organic Chemicals Catalog, 32, 1994, Tokyo Kasei Co., Ltd.).The oxide compounds, per se, may be used as a reagent, or thoseencapsulated in liposomes or the like may also be used. Although theamount of the oxygen source is not particularly limited, preferableamount may be at least 1 μmol or more, preferably 10-30 μmol, and morepreferably about 10-20 μmol based on nitrogen monoxide to be measured.For measurement of samples from a living body, from 10 to 20 μmol of theoxide compound may preferably be added to the samples. However, arequired amount of the oxygen source is generally supplied by dissolvedoxygen. If the amount of oxygen source is extremely small, measuringsensitivity may sometimes be lowered, and if an extremely large amountof oxygen source exists, emission of fluorescence may bedisadvantageously affected. Therefore, it is preferred that an amount ofnitrogen monoxide to be measured is predicted by a preliminaryexperiment or a known method so that the oxygen source within anappropriate concentration range can be applied. The reaction can becarried out at a temperature of from 10° C. to 25° C.

EXAMPLES

[0053] The present invention will be further explained more specificallyby way of the following examples. However, the scope of the presentinvention is not limited to these examples. Synthetic scheme of Example1 is shown below.

Example 1

[0054] Preparation of4-amino-5-(N-methylamino)-2′,7′-difluoro-3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-oneDAF-FM)

[0055] (a) 2,3-Dimethyl-6-nitroacetanilide (1)

[0056] 2,3-Dimethyl-6-nitroaniline (25.3 g, 0.152 mol) was dissolved inacetic acid (290 ml), and the solution was added with acetic anhydride(15 ml, 0.159 mol) and refluxed for 1 hour. After the solvent wasevaporated, the residue was recrystallized from ethanol to obtain thetitle compound (29.3 g, 98%).

[0057]¹H-NMR (300 MHz, CDCl₃) δ2.18 (3H, s); 2.24 (3H, s); 2.39 (3H, s);7.18 (1H, d, J=8.3 Hz); 7.80 (1H, d, J=8.3 Hz); 8.46 (1H, s)

[0058] (b) 3-Acetamide-4-nitrophthalic acid (2)

[0059] 2,3-Dimethyl-6-nitroacetanilide (29.2 g, 0.140 mol) was suspendedin water (1 liter), and the suspension was added with MgSO₄ (100 g,0.831 mol) and refluxed, and then added with KMnO₄ (133 g, 0.841 mol)suspended portionwise in water (2 liters in total). The hot reactionmixture was filtered, and the filtrate was added and saturated withsodium chloride. After cooling, the mixture was acidified with cold HCl,and precipitates were collected by filtration. The filtrate wasextracted with ethyl acetate, and the solvent was evaporated to obtainthe title compound (19.6 g, 52%).

[0060]¹H-NMR (300 MHz, DMSO-d₆) δ1.97 (3H, s); 7.90 (1H, d, J=8.4 Hz);8.04 (1H, d, J=8.4 Hz); 10.13 (1H, s); 13.5 (2H, br)

[0061] (c) 3-Acetamido-4-nitrophthalic anhydride (3)

[0062] 3-Acetamido-4-nitrophthalic acid (0.538 g, 2.00 mmol) wasdissolved in acetic anhydride (10 ml) at 80° C., and the solution wasadded with acetyl chloride (1 ml) and stirred for 2 hours. The solventwas evaporated, and a small amount of anhydrous dichloromethane wasadded to the residue, and precipitates were collected by filtration toobtain the title compound (0.29 g, 58%).

[0063]¹H-NMR (300 MHz, CDCl₃) δ2.35 (3H, s); 7.89 (1H, d, J=8.2Hz); 8.45(1H, d, J=8.2 Hz); 9.01 (1H, s)

[0064] (d)4-Amino-5-nitro-2′,7′-difluoro-3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one(4:3-amino-4-nitrodifluorofluorescein)

[0065] 3-Acetamido-4-nitrophthalic anhydride (1.3 g, 5.0 mmol) and4-fluororesorcinol (1.3 g, 10 mmol) were added to methanesulfonic acid(10 ml), and the mixture was stirred at 80° C. under an argon atmospherefor 60 hours. The reaction mixture was added with water (120 ml) andrefluxed for 2 hours. The reaction mixture was adjusted to pH 2, andprecipitates were collected by filtration. The obtained product wasdried and purified by silica gel chromatography (3% methanol/97%dichloromethane, v/v) to obtain the title compound (1.3 g, 61%).

[0066]¹H-NMR (300 MHz, DMSO-d₆) δ6.40 (d, 1H, J=8.6); 6.87 (d, 2H, J=7.3Hz); 6.89 (d,2H, J=11.3 Hz); 7.95 (br,2H); 8.36 (d, 1H, J=8.6 Hz); 10.8(br, 2H)

[0067] (e) 4,5-Diamino-2′,7′-difluoro-3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-[9H]-xanthen]-3-one(5: 3,4-diaminodifluorofluorescein) (DAF-7)

[0068] 4-Amino-5 -nitro-2′,7′-difluoro-3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one (1.3 g, 3.0 mmol) was addedto an aqueous solution of Na₂S and NaSH, and the mixture was refluxedfor 24 hours. The reaction mixture was cooled, and adjusted to pH 3 withhydrochloric acid, and precipitates were collected by filtration. Theobtained product was dried and purified by silica gel chromatography toobtain the title compound (0.94 g, 78%).

[0069]¹H-NMR (300 MHz, DMSO-d₆) δ5.10 (s, 2H); 5.94 (s, 2H); 6.11 (d,1H, J=7.9 Hz); 6.48 (d, 2H, J=11.3 Hz); 6.81 (d, 1H, J=7.9 Hz); 6.83 (d,2H, J=7.5 Hz); 10.6 (br, 2H)

[0070](f)4-Amino-5-(N-methylamino)-2′,7′-difluoro-3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one(6: DAF-FM)

[0071] DAF-7 (0.94 g, 2.4 mmol) was dissolved in ethanol (200 ml), andthe solution was added with methyl iodide and stirred at 80° C. underargon atmosphere for 3 hours. The product was purified by silica gelchromatography and preparative TLC to obtain the title compound (0.15 g,15%). The compound was further purified by recrystallization frommethanol.

[0072]¹H-NMR (300 MHz, DMSO-d₆) δ2.79 (d, 3H, J=4.6 Hz); 5.32 (q, 1H,J=4.6 Hz); 6.05 (s, 2H); 6.23 (d, 1H, J=7.8 Hz); 6.48 (d, 2H, J=11.3Hz); 6.65 (d, 1H, J=7.8 Hz); 6.83 (d, 2H, J=7.5 Hz); 10.6 (br, 2H)

[0073] MS m/z 412 (M+)

[0074] m.p. 265° C.

[0075] EA for C₂₁H₁₄F₂N₂O₅.CH₄O

[0076] Calcd.: C, 59.46; H, 4.08; N 6.31

[0077] Found: C, 59.45; H, 3.78; N 6.02

[0078](g)4,5-Diamino-3′,6′-bis(acetyloxy)-2′,7′-difluoro-spiro[isobenzofuran-1(3H),9′-[9H]-xanthen]-3-one(7: DAF-FM DA)

[0079] Acetic anhydride (52 ml, 0.55 mmol) was added to DAF-FM (94 mg,0.23 mmol) and Cs₂CO₃ (84 mg, 0.26 mmol) suspended in acetonitrile (20ml), and the mixture was stirred at room temperature for 2 hours. Thereaction mixture was concentrated under reduced pressure, and theresidue was purified by silica gel chromatography (84 mg, 75%). Theproduct was further recrystallized from 2-isopropanol to obtain thetitle compound.

[0080]¹H-NMR (300 MHz, CDCl₃) δ2.34 (s, 6H); 2.93 (s, 3H); 3.40 (br,1H); 5.05 (s, 2H); 6.48 (d, 1H, J=7.9 Hz); 6.73 (d, 2H, J=10.1 Hz); 6.87(d, 1H, J=7.9 Hz); 7.09 (d, 2H, J=6.2 Hz)

[0081] MS m/z 496(M+)

[0082] m.p. 135° C.

[0083] EA for C₂₅H₁₈F₂N₂O₇.C₃H₈O.0.1H₂O

[0084] Calcd.: C, 60.23; H, 4.73; N 5.02

[0085] Found: C, 60.01; H, 4.43; N 5.00

[0086] (h)2″,7″-Difluoro-3″,6″-dihydroxy-spiro[1′-methyltriazolo[4′,5′:4,5]isobenzofuran-1(3H),9″-[9H]xanthen]-3-one(8: DAF-FM T)

[0087] DAF-FM (27 mg, 65 mmol) was dissolved in methanol and bubbledwith nitrogen monoxide gas. The solvent was evaporated, and the residuewas purified by silica gel chromatography (10% methanol/90%dichloromethane v/v, 0.02% acetate, v/v). The obtained product wasdissolved in a small amount of 2 N NaOH aqueous solution, and thesolution was adjusted to pH 3-4 with hydrochloric acid. The precipitateswere collected by filtration and dried to obtain the title compound (13mg, 48%).

[0088]¹H-NMR (300 MHz, acetone-d₆) δ4.50 (s, 3H); 6.69 (d, 2H, J=11.2Hz); 6.94 (d, 2H, J=17.5); 7.41 (d, 1H, J=8.6); 8.25 (d, 1H, J=8.6 Hz);9.6 (br, 2H)

[0089] MS m/z 423 (M+)

[0090] m.p.>300° C.

Example 2

[0091] pH Characteristics of DAF-FM T

[0092] Triazole derivatives prepared by the method disclosed in U.S.Pat. No. 5,874,590 (DAF-2T and DAF-5T, the structures thereof are shownbelow) and DAF-FM T were dissolved at 1 μM in sodium phosphate buffersadjusted to each of the pH values, and fluorescence intensity wasmeasured. The excitation wavelength and fluorescence wavelength were495-515 (nm) for DAF-2T, 505-520 (nm) for DAF-5T and 495-515 (nm) forDAF-FMT, respectively. The results are shown in FIG. 1. As a result, pKaof the hydroxyl group was 6.27 for DAF-2T, 4.59 for DAF-5T, and 4.38 forDAF-FMT, respectively, and pKa of the proton of the triazole ring was7.94 for DAF-2T and 7.41 for DAF-5T. As for DAF-FM T, a dissociableproton was not present in the triazole ring, and therefore the compoundwas not influenced by pKa and it gave a constant fluorescence intensityat pH 5.8 or higher.

Example 3

[0093] Photostability of DAF-FM T

[0094] Photostability of the triazole derivatives produced by the methoddisclosed in U.S. Pat. No. 5,874,590 (DAF-1T, DAF-2T, DAF-4T and DAF-5T,the structures thereof are shown below) and DAF-FM T was tested bydirect irradiation with sunlight. The dyes were each dissolved in 0.1 Msodium phosphate buffer (pH 7.4) at a concentration of 1 μM, and thesolution was put into vials and exposed to direct sunlight under fineweather for a certain period of time. Samples were collected from thesolutions, and fluorescence intensity was measured and compared withrespective initial values. The results are shown in FIG. 2. DAF-FM T ofthe present invention had higher photostability than the other triazolecompounds.

Example 4

[0095] Measurement of Extracellularly Produced Nitrogen Monoxide

[0096] Bovine aorta endothelial cells were cultured, and the cells wereremoved with trypsin and collected by centrifugation, and then suspendedin 2 ml of PBS(+) in which DAF-FM (7 μM) was dissolved. These cells weretransferred to a cell for fluorescence measurement, and fluorescenceintensity was measured (Ex.: 500 nm, Em.: 515 nm) at 37° C. withstirring. The cells were stimulated with a calcium ionophore A23187during the measurement so that the cells generated nitrogen monoxide.The results are shown in FIG. 3. As control experiments, an inhibitor ofnitrogen monoxide synthetase was added after the above measurement, oralternatively, the measurement was performed in the presence of theinhibitor from the beginning. The cease of increase of fluorescence wasobserved, which verified that the increase of fluorescence intensity wascaused by nitrogen monoxide.

Example 5

[0097] Imaging of Intracellular Nitrogen Monoxide

[0098] Bovine aorta endothelial cells were cultured on a dish forimaging. The cell supernatant was replaced with a solution of DAF-FM DA(10 μM) in PBS(+) (containing 0.2% DMSO) to load the dye on the cells at37° C. for about 1 hour. After the cells were washed, the PBS(+) wasreplaced with PBS(+) not containing the dye, and the cells were observedby a fluorescence microscope provided with an excitation filter (490nm), dichronic mirror (505 nm), and barrier filter (515 nm (long pass)).The measurement was performed in an incubator kept at 37° C. Imaginginterval was set to 10 seconds, and images taken by a cooledcharge-coupled device camera were analyzed by an image analyzer. Theresults are shown in FIG. 4. The results shown in the figure areindicated by averages of relative fluorescence intensities of sevenarbitrary cells based on fluorescence intensities at the start of themeasurement obtained. The fluorescence intensity was increased bystimulation with bradykinin, and the increase was suppressed in thepresence of the inhibitor of nitrogen monoxide synthetase, whichindicates that intracellular nitrogen monoxide was successfully imaged.The data of imaged fluorescence intensity were obtained in a similarmanner.

[0099] Industrial Applicability

[0100] The compounds of the present invention are useful as an agent fornitrogen monoxide measurement. The compounds of the formula (I) of thepresent invention have a property of efficiently reacting with nitrogenmonoxide to give the fluorescent compounds of the formula (II). Thecompounds of the formula (II) have characteristic features in that theyemit strong fluorescence by irradiation of excitation light having along wavelength which does not cause damages to living tissues andcells, and that their fluorescence intensity is not substantiallyinfluenced by pH and they have excellent photostability.

What is claimed is: 1.4-Amino-5-(N-methylamino)-2′,7′-difluoro-3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one(6: DAF-FM).
 2. A composition for measurement of nitrogen monoxide,which comprises a compound according to claim
 1. 3. A method formeasuring nitrogen monoxide which comprises (1) reacting the (DAF-FM) ofclaim 1 with nitrogen monoxide, and (2) detecting a compound formed bythe reaction in (1).